Genomes perform several critical tasks to ensure normal development, homeostasis and health. First, gene transcription needs to be accurately regulated and defects in gene regulation can result in human diseases such as cancer and diabetes. Second, chromosomes need to be replicated and then accurately and reliably transmitted to daughter cells. Chromosome segregation requires extensive chromosome compaction. Defects in this process can lead to genome instability, aneuploidy and cancer. Recent studies have revealed that the spatial organization of chromosomes is a major factor in controlling both gene expression and chromosome segregation. The work proposed here will for the first time characterize the three-dimensional (3D) arrangement of chromosomes during different stages of the cell cycle at unprecedented resolution which will allow the identification of cis-elements involved in the 3D folding of chromosomes. A set of novel and powerful genomic technologies (5C and Hi-C) that allow the mapping of 3D chromosome folding will be applied to the study of the human genome. These technologies will be combined with the use of synchronous cell populations and genome-wide analysis of regulatory elements to characterize the different chromosome conformations during the cell cycle and to identify the cis-elements and some trans-factors involved. In addition, the processes that determine 3D folding of the genome, such as transcription and modulation of DNA topology will be studied. This project will provide answers to long-standing questions related to long-range gene regulation, chromosome segregation, the epigenetic transmission of transcription profiles to daughter cells, and the still largely mysterious process of formation of compact metaphase chromosomes.